MUST INQUIRE! ELISafe 19 COVID-19 IgG ELISA Detection Kit
Product#: HM-EL001
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ELISafe 19™ COVID-19 IgG ELISA Detection Kit
Product Code: HM-EL001
Packings: EL001-1KT
An In vitro diagnostic kit for qualitative detection of IgG antibodies against S1 fragment of SARS-CoV-2 by standard ELISA method from human serum/plasma samples.
• Validated and approved by ICMR
• Sensitivity of assay : 99%
• Specificity of assay : 100%
• License to manufacture and sale by CDSCO
• ISO 13485:2016 Certified
• CE marked for IVD (98/79/EC)
1. Technical Features
| Solid phase | S1 spike protein containing the receptor binding domain |
| Conjugate | Anti-human IgG conjugated with HRP |
| Reagents | Ready-to-use except wash buffer concentrate (10X), which need to be diluted. Sample diluent and HRP Conjugate Secondary Ab |
| Sample dilution Controls | Human Serum or Plasma (1: 100 in sample dilution buffer) (E.g. 5µl sample + 495µl sample dilution buffer) Positive control and negative control |
| Method | Indirect ELISA |
| Incubations | Samples – 60 minutes (37°C) HRP conjugate – 45 minutes (37°C) TMB substrate – 20 minutes (Room temperature) |
| Result interpretation | Qualitative NC - Negative control “Negative” < Avg. NC + 0.2 (Cutoff) < “Positive” |
| Measurement | 450nm |
| Kit format | 96-well pre-coated plate |
| Shelf Life | 18 months |
2. Kit contents
| Code | Description | Color code | Quantity | Shipping temperature |
Storage temperature |
|---|---|---|---|---|---|
| EL001A | S1 spike protein SARS-Cov-2 coated 96 well microplate | - | 1 No. | 2 – 8°C | 2 – 8°C |
| EL001B | Plate Sealer | - | 2 Nos. | 2 – 8°C | 15 – 30°C |
| EL001C | Wash Buffer Concentrate 10X | WB | 50ml | 2 – 8°C | 15 – 30°C |
| EL001D | Sample Dilution Buffer 1X | SB | 2 x 50ml | 2 – 8°C | 2 – 8°C |
| EL001E | Sample Diluent Powder | SDP | 2 x 2.5g | 2 – 8°C | 2 – 8°C |
| EL001F | Secondary Antibody Dilution Buffer 1X | Sec Ab DB | 6ml | 2 – 8°C | 2 – 8°C |
| EL001G | HRP Conjugate Secondary Ab | Sec Ab | 20µl | 2 – 8°C | 2 – 8°C |
| EL001H | TMB | TMB | 6ml | 2 – 8°C | 2 – 8°C |
| EL001I | Stop Solution | SS | 5ml | 2 – 8°C | 2 – 8°C |
| EL001J | Negative Control | NC | 5µl | – 20°C | – 20°C |
| EL001K | Positive Control | PC | 5µl | – 20°C | – 20°C |
This kit comprises of 96 tests (including 3 controls in duplicates).
3. Usefulness of the test
3.1. Helps to evaluate immune status in critical patients and patients with over 5 days of onset of symptoms.
3.2. Helps in screening healthcare personnel and identify those who are already immune and can return to work.
This will minimize the risk of spreading the virus to colleagues and other patients.
3.3. Helps in epidemiological studies to study herd immunity.
3.4. Helps to identify IgG immune status of COVID 19 recovered patients so that their plasma can be used for
therapeutics.
3.5. Help in vaccine trials and vaccination follow up when vaccines will be ready.
4. Intended use
4.1. The kit is for in vitro diagnostic use only. The kit is intended for qualitative detection of IgG antibodies against
S1 fragment of SARS-CoV-2 by standard ELISA method from human serum/plasma samples.
4.2. The kit has to be used by trained healthcare professionals.
5. Principle of the Assay
| The ELISafe 19™ kit is developed and manufactured for the qualitative measurement of the anti-COVID-19 IgG antibody in human serum/plasma. In this technique, 1:100 diluted human serum/plasma sample is added to the microwell plate was coated with the S1 recombinant protein of SARS-CoV-2. After incubation, the unbound protein matrix present in sample is removed in the subsequent washing steps. A horseradish peroxidase (HRP) coupled with anti-human IgG is added to each well. After the incubation period, an immunocomplex of “COVID-19 recombinant S1 protein–human anti-COVID-19 IgG antibody-HRP labeled anti-human IgG tracer antibody” is formed if there is specific SARS CoV 2 IgG antibody present in the test sample. |
|
| The unbound antibody is removed in subsequent washing steps. HRP-labeled antibody bound to the microwell is then incubated with a chromogenic substrate solution for 20 minutes for color development followed by stopping the reaction using stop solution. The plate is then read on ELISA plate reader at 450nm wavelength. This kit has been validated on the positive cases of SARS-CoV-2 patients who were tested positive using Real time q-PCR and the appropriate negative serum/plasma samples. |
 |
6. Materials required but not provided
Biosafety laminar hood level II
ELISA plate reader capable of measuring absorbance at 450nm
Micropipettes, multichannel pipettes and tips
Plate washer
Lint free tissue paper
Deionized/distilled water
10% Bleach for discard
ELISA plate reader capable of measuring absorbance at 450nm
Micropipettes, multichannel pipettes and tips
Plate washer
Lint free tissue paper
Deionized/distilled water
10% Bleach for discard
7. Procedure
Warning and Precautions
Wear protective gloves/protective clothing/eye protection/face protection. Follow proper aseptic techniques
while handling the specimens. Standard precautions as per established guidelines should be followed while
handling the clinical specimens.
USERS ARE ADVISED TO REVIEW THE ENTIRE PROCEDURE BEFORE STARTING THE ASSAY.
ELISA PLATE AND REAGENTS SUPPLIED IN THIS KIT ARE FOR SINGLE USE ONLY.
Wear protective gloves/protective clothing/eye protection/face protection. Follow proper aseptic techniques
while handling the specimens. Standard precautions as per established guidelines should be followed while
handling the clinical specimens.
USERS ARE ADVISED TO REVIEW THE ENTIRE PROCEDURE BEFORE STARTING THE ASSAY.
ELISA PLATE AND REAGENTS SUPPLIED IN THIS KIT ARE FOR SINGLE USE ONLY.
| Do’s | Don’ts |
|
|
7.1 Specimen type
Human Serum or Plasma
7.2 Specimen collection and handling
7.2.1. Disinfect the vacutainer (containing suitable anticoagulant for plasma collection)/blood collection tube
(without anticoagulant for collection of serum) by applying 70% alcohol/spirit to the rubber stopper.
7.2.2. Palpate the vein of patient before disinfection of venipuncture site.
7.2.3. Cleanse the site with 70% alcohol/spirit and allow it to dry.
7.2.4. Collect the required volume of blood by venipuncture.
7.2.5. Separate plasma/serum according to standard procedure.
7.2.6. Sterilize the needle, syringe and other materials used for blood collection by autoclaving before discarding.
7.3 Reagent preparation
Prior to use, allow all reagents to attain room temperature (RT). Important: if using strips take out only which is
required, rest keep back at 2-8°C in pouch.
7.3.1. Dilution of wash buffer
Add entire quantity (50ml) of wash buffer concentrate 10X (EL001C) to 450ml distilled/milli Q water to dilute it to
1X working wash buffer. This 1X wash buffer can be stored at room temperature.
7.3.2. Preparation of sample diluent buffer
Add entire quantity of one Sample diluent powder (EL001E) to the respective one bottle of Sample dilution buffer
(EL001D). Swirl the bottle to dissolve the powder completely. (Part EL001E and part EL001D are provided in 2 nos
each). Keep at 37°C for 30 minutes for complete dissolution.
Note: It is recommended to prepare sample dilution buffer just prior to sample dilution.
7.3.3. Sample preparation
Dilute the serum/plasma sample 1:100 using sample dilution buffer prepared in step 7.3.2. Mix well prior to
performing the assay. Diluted samples are not stable and should be used immediately on dilution.
7.3.4. Dilution of negative and positive control
Dilute negative control (EL001J) and positive control (EL001K) 1:100 using sample dilution buffer prepared in
step 7.3.2. Mix well prior to performing the assay.
7.3.5. Dilution of secondary antibody
Add 20μl secondary antibody (EL001G) to 6ml secondary antibody dilution buffer (EL001F).
7.4 Assay procedure
7.4.1. Remove the plate sealer and wash all the wells once with 1X wash buffer.
7.4.2. Designate the wells with “BLANK”, “POSITIVE CONTROL”, “NEGATIVE CONTROL” and “SAMPLE IDs””
7.4.3. Add 50μl sample diluent buffer in BLANK.
7.4.4. Add 50μl each diluted positive and negative controls and diluted samples to respective wells.
7.4.5. Agitate/tap gently from all the sides of the plate so that samples are spread homogenously. Avoid air
bubbles if any. Cover the plate with plate sealer (EL001B). Incubate the plate at 37°C for 1 hour.
7.4.6. Aspirate and discard the contents from each well and add 300μl of diluted wash buffer to remove unbound
IgG. Swirl the plate and discard the contents. Alternatively plate washer can be used for washing the wells
more effectively.
7.4.7. Repeat washing step 3 times.
7.4.8. Add 50μl of diluted HRP conjugate secondary antibody to each well.
7.4.9. Incubate at 37°C for 45 minutes.
7.4.10. Discard the HRP conjugate secondary Ab from each well and add 300μl of diluted wash buffer to remove
unbound IgG. Swirl the plate and discard the contents.
7.4.11. Repeat the washing step 6 times.
7.4.12. Pat the plate dry on lint free tissue paper.
7.4.13. Add 50μl of TMB Solution (EL001H) to each well and incubate at room temperature for 20 minutes.
Positive samples will give blue color.
7.4.14. Add 25μl stop solution (EL001I) to each well. Incubate at room temperature for 5 minutes.
7.4.15. Mix gently by tapping the plate and read absorbance on plate reader at 450nm.
Human Serum or Plasma
7.2 Specimen collection and handling
7.2.1. Disinfect the vacutainer (containing suitable anticoagulant for plasma collection)/blood collection tube
(without anticoagulant for collection of serum) by applying 70% alcohol/spirit to the rubber stopper.
7.2.2. Palpate the vein of patient before disinfection of venipuncture site.
7.2.3. Cleanse the site with 70% alcohol/spirit and allow it to dry.
7.2.4. Collect the required volume of blood by venipuncture.
7.2.5. Separate plasma/serum according to standard procedure.
7.2.6. Sterilize the needle, syringe and other materials used for blood collection by autoclaving before discarding.
7.3 Reagent preparation
Prior to use, allow all reagents to attain room temperature (RT). Important: if using strips take out only which is
required, rest keep back at 2-8°C in pouch.
7.3.1. Dilution of wash buffer
Add entire quantity (50ml) of wash buffer concentrate 10X (EL001C) to 450ml distilled/milli Q water to dilute it to
1X working wash buffer. This 1X wash buffer can be stored at room temperature.
7.3.2. Preparation of sample diluent buffer
Add entire quantity of one Sample diluent powder (EL001E) to the respective one bottle of Sample dilution buffer
(EL001D). Swirl the bottle to dissolve the powder completely. (Part EL001E and part EL001D are provided in 2 nos
each). Keep at 37°C for 30 minutes for complete dissolution.
Note: It is recommended to prepare sample dilution buffer just prior to sample dilution.
7.3.3. Sample preparation
Dilute the serum/plasma sample 1:100 using sample dilution buffer prepared in step 7.3.2. Mix well prior to
performing the assay. Diluted samples are not stable and should be used immediately on dilution.
7.3.4. Dilution of negative and positive control
Dilute negative control (EL001J) and positive control (EL001K) 1:100 using sample dilution buffer prepared in
step 7.3.2. Mix well prior to performing the assay.
7.3.5. Dilution of secondary antibody
Add 20μl secondary antibody (EL001G) to 6ml secondary antibody dilution buffer (EL001F).
7.4 Assay procedure
7.4.1. Remove the plate sealer and wash all the wells once with 1X wash buffer.
7.4.2. Designate the wells with “BLANK”, “POSITIVE CONTROL”, “NEGATIVE CONTROL” and “SAMPLE IDs””
7.4.3. Add 50μl sample diluent buffer in BLANK.
7.4.4. Add 50μl each diluted positive and negative controls and diluted samples to respective wells.
7.4.5. Agitate/tap gently from all the sides of the plate so that samples are spread homogenously. Avoid air
bubbles if any. Cover the plate with plate sealer (EL001B). Incubate the plate at 37°C for 1 hour.
7.4.6. Aspirate and discard the contents from each well and add 300μl of diluted wash buffer to remove unbound
IgG. Swirl the plate and discard the contents. Alternatively plate washer can be used for washing the wells
more effectively.
7.4.7. Repeat washing step 3 times.
7.4.8. Add 50μl of diluted HRP conjugate secondary antibody to each well.
7.4.9. Incubate at 37°C for 45 minutes.
7.4.10. Discard the HRP conjugate secondary Ab from each well and add 300μl of diluted wash buffer to remove
unbound IgG. Swirl the plate and discard the contents.
7.4.11. Repeat the washing step 6 times.
7.4.12. Pat the plate dry on lint free tissue paper.
7.4.13. Add 50μl of TMB Solution (EL001H) to each well and incubate at room temperature for 20 minutes.
Positive samples will give blue color.
7.4.14. Add 25μl stop solution (EL001I) to each well. Incubate at room temperature for 5 minutes.
7.4.15. Mix gently by tapping the plate and read absorbance on plate reader at 450nm.
| Important Guidelines 1. Accuracy of the assay depends on pipetting skills of the personnel. Inappropriate addition and mixing
practices may result in erroneous and false-positive or false-negative results. 2. Use of a repeating pipettor is recommended to deliver the reagents to the wells. This saves time and helps maintain more precise incubation times. 3. Pipette tip should be equilibrated with the reagent before use. This is carried out by slowly filling up the tip with reagent and gently expelling the contents, several times. 4. To obtain statistically significant data, it is important to assay all the samples in duplicates. 5. All reagents should be mixed gently and thoroughly prior to use. Avoid foaming. 6. Avoid air bubbles in the wells as this can result in lower binding efficiency and higher CV% of duplicate reading. |
| PROCEDURE AT GLANCE | |||||
| Step | Action | Volume per well |
Incubation Time |
Incubation Temperature |
Repeat |
| 1 | Plate wash with 1X washing buffer | 300µl | NA | NA | Once |
| 2 | Addition of blank, controls and samples | 50µl | 60 minutes | 37°C | NA |
| 3 | Removal of samples | - | - | - | - |
| 4 | Plate wash with 1X washing buffer | 300µl | NA | NA | 3 times |
| 5 | Addition of diluted HRP Conjugate Secondary Ab | 50µl | 45 minutes | 37°C | NA |
| 6 | Removal of antibody | - | - | - | - |
| 7 | Plate wash with 1X washing buffer | 300µl | NA | NA | 6 times |
| 8 | Addition of TMB Solution | 50µl | 20 minutes | Room temperature |
- |
| 9 | Addition of Stop Solution | 25µl | 5 minutes | Room temperature |
- |
| 10 | Measurement of OD at 450nm on ELISA plate reader | - | - | - | - |
8. Data analysis
8.1. “Positive control” and “Negative control” work as markers of kit performance.
8.2. Calculate P/N ratio of Positive control which is defined as the ratio of average OD value of Positive control
and average OD of Negative control.
8.2. Calculate P/N ratio of Positive control which is defined as the ratio of average OD value of Positive control
and average OD of Negative control.
The test is considered to be valid if P/N ratio of Positive control is greater than 1.5
8.3. Calculate cutoff value using formula as given below,
Cutoff calculations = Avg. NC + 0.2
Interpretation of the results:
1) For unknown sample if O.D value > Cutoff value, then the sample should be considered as “Positive”.
2) For unknown sample if O.D value < Cutoff value, then the sample should be considered as “Negative”
9. Limitations of the assay
9.1. This kit is intended only for qualitative detection of SARS-CoV-2 IgG. Test results should not be used as
the sole basis to diagnose or exclude SARS-CoV-2 infection or to confirm infection status. Confirmation of
infection must be correlated with clinical symptoms and other tests.
9.2. There is a possibility of obtaining False-negative result if
- Blood samples are collected in the early phase of onset of infection (within one week after infection onset)
- Patients/suspects having low innate immunity or if using drugs that supress immune function.
9.3. There is a possibility of obtaining False-positive result if
- Suspect/patient is previously infected with SARS or other strains of Coronavirus or who have sticky
serum/plasma or autoimmune diseased patients.
9.4. Bacterial or fungal contamination of serum specimens or reagents, or cross-contamination between
reagents may cause erroneous results.
the sole basis to diagnose or exclude SARS-CoV-2 infection or to confirm infection status. Confirmation of
infection must be correlated with clinical symptoms and other tests.
9.2. There is a possibility of obtaining False-negative result if
- Blood samples are collected in the early phase of onset of infection (within one week after infection onset)
- Patients/suspects having low innate immunity or if using drugs that supress immune function.
9.3. There is a possibility of obtaining False-positive result if
- Suspect/patient is previously infected with SARS or other strains of Coronavirus or who have sticky
serum/plasma or autoimmune diseased patients.
9.4. Bacterial or fungal contamination of serum specimens or reagents, or cross-contamination between
reagents may cause erroneous results.
10. Performance characteristics
10.1. Sensitivity *
Ninety Five (95) positive serum samples were collected from individuals two weeks after they tested positive by
RT-PCR and they were analyzed by ELISafe 19™. Out of that Ninety Four (94) samples showed positive result.
Ninety Five (95) positive serum samples were collected from individuals two weeks after they tested positive by
RT-PCR and they were analyzed by ELISafe 19™. Out of that Ninety Four (94) samples showed positive result.
Sensitivity of the assay = 99%
10.2. Specificity * A total of 336 samples obtained before outbreak of SARS-CoV-2 (before January 2020) were tested using ELISafe
19™. None of the samples were detected false positive.
Specificity of the assay = 100%
11. Storage and shelf life
11.1 Store all the reagents at temperatures mentioned on the product label.
11.2 Store away from direct heat and bright light.
11.3 Shelf life of the product is 18 months.
11.2 Store away from direct heat and bright light.
11.3 Shelf life of the product is 18 months.
12. Plate template
The plate template mentioned below can be used for ease of setting up the assay.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | BL | BL | S11 | S19 | S27 | S35 | S43 | S51 | S59 | S67 | S75 | S83 |
| B | NC | NC | S12 | S20 | S28 | S36 | S44 | S52 | S60 | S68 | S76 | S84 |
| C | PC | PC | S13 | S21 | S29 | S37 | S45 | S53 | S61 | S69 | S77 | S85 |
| D | S1 | S6 | S14 | S22 | S30 | S38 | S46 | S54 | S62 | S70 | S78 | S86 |
| E | S2 | S7 | S15 | S23 | S31 | S39 | S47 | S55 | S63 | S71 | S79 | S87 |
| F | S3 | S8 | S16 | S24 | S32 | S40 | S48 | S56 | S64 | S72 | S80 | S88 |
| G | S4 | S9 | S17 | S25 | S33 | S41 | S49 | S57 | S65 | S73 | S81 | S89 |
| H | S5 | S10 | S18 | S26 | S34 | S42 | S50 | S58 | S66 | S74 | S82 | S90 |
13. Troubleshooting
| Problem | Cause | Solution |
| Poor reproducibility of test | Inappropriate washing/aspiration | Make sure that the plate washer is programmed properly and contents are completely aspirated from the wells |
| Wells are scratched with pipette/handling needles |
Aspirate and dispense the solutions from and into the wells along the walls. DO NOT touch the pipette tip to the well surface |
|
| Samples contain particulate matter | Clarify the test samples by centrifugation prior to use | |
| Weak/no signal | OD taken at wrong wavelength | Read OD values at 450nm |
| Strips not washed before sample addition or incorrect volumes of the reagents added |
Wash the plate once with wash buffer before sample addition or repeat the assay with correct volumes as mentioned in the technical datasheet |
|
| Insufficient incubation | Incubate the plate at exact temperature and duration as mentioned in the technical datasheet |
|
| High OD value of Negative control | Same pipettes used for Positive and Negative controls |
Change the micropipette tips while addition of Negative/Positive control |
| High background | Insufficient washing | Make sure that the wells are washed thoroughly |
| Liquid substrate not properly protected from light |
Incubate the plate in dark after adding TMB substrate | |
| Contamination of liquid substrate | Check OD value of substrate blank |
